The Biology and Biochemistry of PhoH2 proteins

PhoH2 proteins are found in a diverse range of organisms. To date little is known about these proteins and the role they play in the organisms in which they reside. PhoH2 is a PIN PhoH domain fusion, and these proteins are currently annotated as having unknown function and are described as PhoH like. PhoH domains are thought to be ATPases and all characterised PIN domain proteins are RNases. Most efforts have focussed on determining the role of PIN domain proteins that comprise the toxic component (VapC) of VapBC toxin antitoxin systems, in which the PIN domain is coexpressed as part of an operon with an inhibitor (VapB). In the remaining cases where PIN domain proteins can be found such as PIN PhoH domain fusions, these cases remain unexamined. This thesis describes the biological and biochemical characterisation of the PIN PhoH protein, PhoH2 from Mycobacterium tuberculosis and Mycobacterium smegmatis, along with a preliminary structural characterisation of a thermophilic PhoH2 protein homologue. The phoH2 gene from both mycobacterial organisms was found to be expressed as part of a long mRNA transcript. Examination of these transcripts revealed possible alternative 5’ start sites out of frame with the phoH2 gene. For protein overexpression, and ‘normal’ growth and colony formation with conditional overexpression, phoH2 from M. tuberculosis required 152 bp of the 5’ sequence directly upstream of the annotated phoH2 gene (annotated here as phoH2alt). PhoH2 proteins: PhoH2alt MTB, PhoH2alt MSMEG and PhoH2MSMEG show ATP/Mg²⁺ dependent, sequence specific RNA unwinding and cleavage. The sequence (A C) (A/U) (A/U) (G/C) U was deduced as a substrate for PhoH2, and PhoH2alt MTB also demonstrated unwinding and cleavage activity on its upstream ~152 base RNA transcript, suggesting a potential autoregulatory mechanism. Structural analysis of a thermophilic PhoH2 protein homologue has provided preliminary crystallographic data which along with electron microscopy suggest a ring like hexameric PhoH2 oligomer

Post-transcriptional modulation of the SigF regulon inMycobacterium smegmatisby the PhoH2 toxin-antitoxin

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc²155 and mc²155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc²155 and M. smegmatis mc²155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis

Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophos­phate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, which can then be synthesized and experimentally characterized. Through ASR a novel PDC was generated, designated ANC27, that shares only 78% amino-acid sequence identity with its closest extant homologue (Komagataeibacter medellinensis PDC, GenBank accession No. WP_014105323.1), yet is fully functional. Crystals of this PDC diffracted to 3.5 Å resolution. The data were merged in space group P3221, with unit-cell parameters a = b = 108.33, c = 322.65 Å, and contained two dimers (two tetramer halves) in the asymmetric unit. The structure was solved by molecular replacement using PDB entry 2wvg as a model, and the final R values were Rwork = 0.246 (0.3671 in the highest resolution bin) and Rfree = 0.319 (0.4482 in the highest resolution bin). Comparison with extant bacterial PDCs supports the previously observed correlation between decreased tetramer interface area (and number of interactions) and decreased thermostability

Crystal structure of pyruvate decarboxylase from Zymobacter palmae

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg²⁺ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rᵣ.ᵢ.ₘ. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwₒᵣₖ = 0.186 (0.271 in the highest resolution bin) and Rfᵣₑₑ = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions